Antibodies for cancer therapy and diagnosis

ABSTRACT

The present application describes a method for making antibodies which can be used for cancer diagnosis or therapy. The application also discloses a method for identifying an antigen which is differentially expressed on the surface of two or more distinct cell populations. The application additionally describes human antibodies directed against decay accelerating factor (DAF), as well as therapeutic compositions comprising such antibodies. Moreover, the application discloses a method of treating lung cancer with antibodies directed against DAF.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S.application Ser. No. 10/447,331, filed on May 28, 2003, which is adivisional application of Ser. No. 09/515,825, filed on Feb. 29, 2000,incorporated herein by reference, and to which priority is claimed under35 U.S.C. § 120, which co-pending application is a non-provisionalapplication filed under 37 C.F.R. 1.53(b)(1) which claims priority under35 U.S.C. 119(e) to provisional application No. 60/122,262 filed Mar. 1,1999, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns a method for making antibodies which can,for example, be used for cancer diagnosis or therapy. The inventionfurther provides a method for identifying an antigen which isdifferentially expressed on the surface of distinct cell populations.The present invention additionally provides human antibodies directedagainst decay accelerating factor (DAF), as well as therapeuticcompositions comprising such antibodies. Moreover, the inventionpertains to a method of treating lung cancer with antibodies directedagainst DAF.

2. Description of Related Art

The demonstration of significant anti-tumor efficacy of antibodies haslong been sought-after in the clinic and recently obtained using “naked”chimeric/humanized antibodies (Riethmüller et al., Lancet, 343:1177-1183 (1994); Riethmüller et al., J. Clin. Oncol., 16: 1788-1794(1998); Maloney. et al., Blood, 90: 2188-2195 (1997); McLaughlin et al.,J. Clin. Oncol., 16: 2825-2833 (1998); and Baselga et al., J. Clin.Oncol., 14: 697-699 (1996)) antibodies as well as with radiolabeledmurine antibodies (Press et al., N. Engl. J. Med., 329: 1219-1224(1993); Press et al., Lancet, 346: 336-340, (1995); Kaminski et al., N.Engl. J. Med., 329: 459-465 (1993); Kaminski et al., J. Clin. Oncol.,14: 1974-1981 (1996)). Indeed a chimeric anti-CD20 antibody (Reff etal., Blood, 83: 435-445 (1994)) and a chimeric/humanized anti-HER2antibody (Carter et al. PNAS (USA) 89:4285-4289 (1992)) have recentlybeen approved by US Federal Drug Administration for the treatment ofnon-Hodgkin's lymphoma and metastatic breast cancer, respectively. Thesesuccesses with anti-tumor antibodies in patients has led to renewedinterest in the identification of novel tumor-associated antigenssuitable for antibody targeting.

The traditional approach to obtaining tumor-specific antibodies has beento immunize mice with tumor cells and to screen the resultant monoclonalantibodies for their binding specificity. Unfortunately tumor-bindingantibodies obtained in this way often cross-react with many normalcells, which may interfere with their clinical utility. Ideally onewould like to select rather than screen for antibodies that bindselectively to tumor. The advent of antibody fragment display on phage(McCafferty et al., Nature, 348: 552-554 (1990)) and the development oflarge (>10¹⁰ clone) phage display libraries (Griffiths et al., EMBO J.,13:3245-3260 (1994), Vaughan et al. Nat. Biotechnol. 14: 309-314 (1996))offers a potential way of making antibodies. With antibody phagescreening, unlike hybridoma technology, it is readily possible to obtainantibodies binding antigens that are highly conserved between mouse andman (Nissim et al., EMBO J., 13:692-698 (1994)).

Naïve antibody phage libraries have proved to be a rapid and generalmethod for identifying antibodies binding to purified antigens(Griffiths et al., EMBO J., 13:3245-3260 (1994); Vaughan et al. Nat.Biotechnol. 14: 309-314 (1996); Nissim et al., EMBO J., 13:692-698(1994)). In contrast, panning cellular targets with antibody phage hasproved much more difficult because of the much lower effective antigenconcentration, greater antigen complexity and the tendency of phage tobind non-specifically to cells. Nevertheless, antibodies against cellsurface antigens have been identified (Marks et al., Bio/Technol.,11145-1149 (1992); Portolano et al., J. Immunol., 151:2839-2851 (1993);de Kruif et al, Proc. Natl. Acad. Sci. USA, 92:3938-3942 (1995); VanEwijk et al., Proc. Natl. Acad. Sci. USA, 94:3903-3908 (1997); Cai etal, Proc. Natl. Acad. Sci. USA, 92:6537-6541 (1995); Cai et al, Proc.Natl. Acad. Sci. USA, 93:6280-6285 (1996); Cai et al., Proc. Natl. Acad.Sci. USA, 94:9261-9266 (1997)). Melanoma specific antibodies have beenidentified by selecting for antibody phage that bind to melanoma cellsbut not melanocytes using antibody phage libraries constructed fromhuman donors immunized with their own tumor cells (Cai et al, Proc.Natl. Acad. Sci. USA, 92:6537-6541 (1995); Cai et al Proc. Natl. Acad.Sci. USA, 93:6280-6285 (1996); Cai et al., Proc. Natl. Acad. Sci. USA,94:9261-9266 (1997)).

Decay Accelerating Factor (DAF), is a GPI-anchored protein that actstogether with two other GPI-anchored proteins, CD46 and CD59, inprotecting host cells from complement-mediated cell lysis(Nicholson-Weller et al. J. Lab. Clin. Med., 123:485-491 (1994)). DAF isexpressed at widely varying levels on tumor cell lines and itsoverexpression correlates with enhanced resistance tocomplement-mediated cell lysis in vitro (Cheung et al., J. Clin.Invest., 81:1122-1128 (1988)). DAF overexpression has been observed on avariety of human tumor tissues including 6/9 lung adenocarcinomas and2/7 lung squamous cell carcinomas (Niehans et al., Am. J. Path.,149:129-142 (1996)). Regarding normal lung tissue, DAF has been detectedby immunohistochemistry on the alveolar epithelium, interstitium andendothelium as well as the bronchial epithelium, glands and ducts plusblood vessels (Niehans et al., Am. J. Path., 149:129-142 (1996)).

Other publications relating to DAF include Hara et al. ImmunologyLetters 37:145-152 (1993); Nicholson-Weller and Wang J. Lab. Clin. Med.123(4):485-491 (1994); Lublin et al. J. Immunol. 137:1629-1635 (1986);WO99/43800; WO98/39659; U.S. Pat. No. 5,695,945; U.S. Pat. No.5,763,224; and WO 86/07062.

Vollmers et al. Cancer Research 49: 2471-2476 (1989); and Vollmers etal. Cancer 76(4): 550-558 (1995) describe the human IgM monoclonalantibody “SC-1” which is said inhibit growth of stomach adenocarcinomacells in vitro and in vivo by inducing apoptosis. Vollmers et al.Oncology Reports 5:549-552 (1998) reports the results of a clinicaltrial in which patients with poorly differentiated stomachadenocarcinoma were treated with the SC-1 antibody. The laterpublication, Hensel et al. Cancer Research 59:5299-5306 (1999),identifies DAF as the antigen bound by SC-1.

SUMMARY OF THE INVENTION

In the present application, a large naïve antibody phage library wasused to search for cancer-associated antigens, thus obviating the needfor creating custom libraries from immunized donors. In addition,antibodies were selected using live rather than fixed cells, to obtainantibodies primarily against native rather than denatured antigens. Thiswas done to facilitate subsequent expression cloning of correspondingantigen as well as enhance the therapeutic potential of antibodiesobtained. Indeed an antigen corresponding to a scFv fragment identifiedwith significant tumor selectivity was cloned according to the presentmethods.

Accordingly, the invention provides a method for making an antibodycomprising the following steps: (a) binding antibody phage from a naïveantibody phage library to a live cancer cell; (b) selecting an antibodyphage or antibody which binds selectively to the live cancer cell; and(c) identifying an antigen to which the antibody phage or antibodybinds.

The invention further provides an antibody derived according to themethod of the preceding paragraph and optionally including amino acidsequence alterations (e.g. additions, deletions and/or substitutions)compared to the antibody selected in step (b)). Moreover, the inventionprovides a method for detecting the antigen comprising exposing a samplesuspected of containing the antigen to the antibody or altered antibodyand determining binding of the antibody or altered antibody to thesample. The invention further provides a method for treating a mammalhaving a disease or disorder comprising administering the above antibodyor altered antibody to the mammal in an amount effective to treat thedisease or disorder.

The invention further provides a method for identifying an antigen whichis differentially expressed on the surface of two or more distinct cellpopulations, comprising the following steps: (a) binding antibody phagefrom a naïve antibody phage library to a first cell population; (b)binding the antibody phage to a second cell population which is distinctfrom the first cell population; (c) selecting an antibody phage orantibody which binds selectively to the first cell population; and (d)identifying an antigen to which the antibody phage or antibody in (c)binds.

The invention further provides an antagonist, such as an antibody,directed against an antigen, wherein the antigen has been identifiedaccording to the method of the previous paragraph.

The invention additionally relates to an isolated human antibody whichis directed against, or specifically binds to, human decay acceleratingfactor (DAF), obtainable by the methods herein. The invention furtherprovides a human antibody which has better binding affinity for DAF thanthe human IgM SC-1 antibody has for DAF, e.g. about 10 nM or betterbinding affinity for human DAF (for instance, in the range from about 1nM to about 1 pM). An example of an antibody with such strong bindingaffinity for DAF is the LU30 antibody herein which has a bindingaffinity (K_(d)) for DAF of about 13 nM as determined using a BIACORE™instrument. The antibody optionally binds an epitope on DAF bound by theLU30, LU13 or LU20 antibodies herein disclosed. The human antibody may,for instance, comprise antigen-binding amino acid residues of the LU30,LU13 or LU20 antibodies. The application additionally provides the humanantibodies designated LU30, LU13 and LU20 herein as well as variants ofany one of those antibodies. Preferred amino acid sequence variantscomprise VH and VL domains which together share about 90-100%, andpreferably about 95-100%, and most preferably 98-100%, amino acidsequence identity with the VH and VL amino acid sequences of the LU30,LU13 or LU20 antibodies as depicted in FIGS. 5A and 5B herein. Onepreferred amino acid sequence variant is an affinity matured variant,which comprises one or more amino acid sequence modifications (e.g.about 1-20, and most preferably about 3-10 amino acid substitutions) inone or more hypervariable regions of the LU30, LU13 or LU20 VH and/or VLamino acid sequences disclosed herein. Another type of variant is aglycosylation variant which has altered glycosylation compared to aparent antibody and thus may have altered effector function(s). While Fvfragment forms (e.g. single chain Fv fragments, scFv) of the LU30, LU13or LU20 antibodies may be used, the variable regions of these antibodiesare optionally fused to heterologous polypeptide(s) such as (1) a toxinpolypeptide(s) to generate an immunotoxin or (2) antibody constantregion sequences to make larger antibody molecules, such as Fabfragments, F(ab′)₂ fragments or intact antibodies. Such intactantibodies generally have human heavy and light chain constant regionsand, therefore, have antibody effector functions, such asantibody-dependent cell-mediated cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC).

In another embodiment, the invention pertains to a pharmaceuticalcomposition comprising a human antibody directed against DAF and apharmaceutically acceptable carrier. In addition, the invention providesan article of manufacture comprising the pharmaceutical composition anda package insert instructing the user of the composition to treat apatient having, or predisposed to, lung cancer with the composition. Thelung cancer to be treated includes small-cell lung cancer, non-smallcell lung cancer, large cell lung carcinoma, lung adenocarcinoma, andsquamous cell lung carcinoma.

In yet a further embodiment, the invention relates to method of treatinglung cancer comprising administering a therapeutically effective amountof an antibody directed against decay accelerating factor (DAF) to ahuman patient. Candidates for treatment with the anti-DAF antibody areoptionally screened to determine DAF expression by tumor cells. Forinstance, DAF overexpression, and/or expression of a DAF glycoform, bythe tumor may be assessed using diagnostic procedures available in theart, such as immunohistochemistry (IHC) or a DNA-based assay (e.g.fluorescent in situ hybridization, FISH). This way, a subpopulation ofcancer patients (e.g. DAF-overexpressing patients or patients expressinga cancer-related variant of DAF) may be identified and those patientscan be treated as described herein. The antibody may be administered inthe neoadjuvant, adjuvant or metastatic settings. Moreover, the antibodyused for such therapy may be conjugated with a cytotoxic agent (examplesof which are provided below) in order to generate an immunotoxin.Preferably, the antibody is a human antibody (e.g. one which has abinding affinity for DAF of about 10 nM or better). The antibody forsuch therapy optionally binds an epitope on DAF bound by any one of theLU30, LU13, LU20, 791T36 or SC-1 antibodies. The antibody for therapymay, therefore, comprise antigen-binding amino acid residues of theLU30, LU13, LU20, 791T36 or SC-1 antibodies. The patient may optionallybe treated with a second different cytotoxic agent, wherein the secondcytotoxic agent is therapeutically effective against lung cancer.Examples of such second cytotoxic agents include, but are not limitedto, navelbine, gemcitabine, a taxoid, carboplatin, cisplatin, etoposide,cyclosphosphamide, mitomycin, vinblastine, an anti-ErbB2 antibody (e.g.HERCEPTIN®, sold by Genentech, Inc., South San Francisco), ananti-angiogenic factor antibody (e.g. an anti-VEGF antibody), ananti-mucin antibody, or a second antibody directed against a differentepitope on DAF. Such therapy with the combination of the antibody andthe second cytotoxic agent may result in a synergistic therapeuticeffect against lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts flow cytometric analysis of phage populations from rounds1, 2 and 3 binding to tumor cell line 1264 (dark) used for selection andnon-tumor cell line BEAS-2B (light) used for counter-selection. Alsoshown is a negative control phage population.

FIGS. 2A-C depict dendrograms for tumor-selective scFv satisfyingprimary and secondary selection criteria (Table 1). Comparisons weremade between scFv amino acid sequences (FIG. 2A) as well as theircomponent V_(H) domains (FIG. 2B), and V_(L) domains (FIG. 2C).

FIG. 3 shows flow cytometric analysis of scFv fragments with tumor(1264, A549, CALU6 and SKLU1) and non-tumor (BEAS-2B and NHEK) celllines.

FIG. 4 shows binding of LU30 scFv (3 μg/ml) to 1264 cells in the absenceand presence of recombinant human DAF (30 μg/ml).

FIGS. 5A and 5B depict the amino acid sequences of the variable light(VL) (FIG. 5A; SEQ ID NOS: 1-3, respectively) and variable heavy (VH)(FIG. 58; SEQ ID NOS:4-6, respectively) domains of human antibodiesLU30, LU13 and LU20 identified in Example 1. Complementarity DeterminingRegion (CDR) residues are those residues in bold and hypervariable loopresidues are within brackets.

TABLE 1 Primary screening of scFv phage clones # tumor clone identityBstNI fingerprint type selective clones^(a) (# clones sequenced) 1 110LU4 (8) 2 49 LU1 (7) 3 10 LU20 (9) 4 7 LU13 (3), LU34 (4)^(b) 5 4 LU22(4) 6 3 LU36 (3) 7 3 LU41 (3) 8 3 LU57 (2) 9 3 LU3 (1), LU77 (2)^(b) 102 LU30 (2) 11 1 LU7 (1) 12 1 LU71 (1) 13 1 LU100 (1) 14 1 LU60 (1)^(c)15 1 LU78 (1) ^(a)Tumor selective clones by phage ELISA: robust bindingto 1264 cells (A₄₅₀-A₆₅₀ ≧ 0.3) and much weaker binding to BEAS-2B cells(≧10-fold lower signal), as judged by phage ELISA. ^(b)Clones LU13 andLU34 are predicted from their nucleotide sequences to generate identicalfingerprint patterns, whereas clones LU3 and LU77 share closely relatedfingerprints that were not distinguishable by our electrophoreticanalysis. ^(c)Codon 3 in V_(H) is amber (TAG) that will be read throughas glutamine in the supE E. coli strain, TG1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The term “antibody” is used in the broadest sense and specificallycovers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; “linear antibodies” (U.S. Pat. No. 5,641,870);single-chain antibody molecules such as single chain Fv fragments(scFv); and multispecific antibodies formed from antibody fragments.

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variants thereof. Preferably, the intactantibody has one or more effector functions.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1 (including human A and non-A allotypes), IgG2,IgG3, IgG4, IgA, and IgA2. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcgammaRIII only, whereas monocytes express FcgammaRI,FcgammaRII and FcgammaRIII. FcR expression on hematopoietic cells issummarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immol9:457-92 (1991). To assess ADCC activity of a molecule of interest, anin vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362or 5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et at. PNAS (USA) 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refer to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

The “antigen-binding” amino acid residues of an antibody are thoseresidues which contact antigen and result in specific binding of theantibody to that antigen. Generally, the antigen-binding residuescoincide with the hypervariable region residues of an antibody. Thehypervariable regions generally comprise amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art. In the preferred embodiment, the human antibody isselected from a phage library, where that phage library expresses humanantibodies (Vaughan et al. Nature Biotechnology 14:309-314 (1996):Sheets et al. PNAS (USA) 95:6157-6162 (1998)); Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991);and Example 1 herein). Human antibodies can also be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol 13:65-93 (1995). Alternatively, the humanantibody may be prepared via immortalization of human B lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes may be recovered from an individual or may have beenimmunized in vitro); see, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147 (1):86-95 (1991); U.S. Pat. No. 5,750,373.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementaritydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit having the desired specificity, affinity, and capacity. In someinstances, Fv framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues which are found neither in therecipient antibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and maximize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522-525 (1986); Reichmannet al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992). The humanized antibody includes a PRIMATIZED™ antibodywherein the antigen-binding region of the antibody is derived from anantibody produced by immunizing macaque monkeys with the antigen ofinterest.

“Single chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). Preferred affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by V_(H) and V_(L) domain shuffling. Random mutagenesis ofCDR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol.226:889-896 (1992).

An antibody which is “directed against” or which “specifically binds to”an antigen of interest, e.g. DAF antigen, is one capable of binding thatantigen with sufficient affinity such that the antibody is useful as atherapeutic agent in targeting the antigen. The antigen here is normallyDAF as it exists in a patient to be treated with the antibody(especially the antigen expressed by tumor cells in the patient).Notwithstanding this, various forms of DAF (e.g. native, recombinant,and synthetic DAF, including DAF variants and fragments) may be used togenerate or raise the antibody.

The “binding affinity” of an antibody for a target antigen, such as DAF,may be determined by equilibrium methods (e.g. enzyme-linkedimmunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics(e.g. BIACORE™ analysis; see Example 1 below), for example.

To determine whether an antibody binds to an “epitope” on an antigen,such as DAF, bound by another antibody, a routine cross-blocking assaysuch as that described in Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Ed Harlow and David Lane (1988), can be performed.

The term “antibody phage” refers to a bacteriophage with an antibody(particularly an antibody fragment such as a scFv, diabody, linearantibody or Fab) displayed on the surface thereof.

A “naïve antibody phage library” comprises a plurality of antibodyphages which have not been derived from an immunized host, i.e. a“non-immunized” phage display library (see, e.g. Vaughan et al. Nat.Biotechnol. 14: 309-314 (1996); and Sheets et al. PNAS (USA)95:6157-6162 (1988)). Exemplary methods for generating such “naïve” or“non-immunized” phage libraries are elaborated herein.

The act of “binding” antibody phage to a cell or cell population entailsexposing or contacting the antibody phage to/with the cell or cellpopulation under appropriate conditions and for a sufficient period oftime such that the antibody displayed on the surface of the phagenoncovalently binds to one or more antigens on the cell or cellpopulation. Generally, those antigen(s) to which the antibody bind(s)are present at the surface of a cell (i.e. are “cell surfaceantigen(s)”). The “antigen” is generally a protein, but may be anon-protein molecule such as a lipid, carbohydrate, glycolipid, nucleicacid etc.

A “live” cell is one which has not been histologically fixed with afixative such as glutaraldehyde. The live cell may be a “primary” cellwhich has, e.g., been surgically removed from a mammal or a “cell line”capable of being continuously cultivated in cell culture. A “live cancercell” is a cancer or tumor cell which has not been histologically fixedand a “live non-cancer cell” is a noncancerous cell (i.e. one which hasnot been derived from a cancer or tumor) which has not beenhistologically fixed.

A “distinct” cell or cell population is one which is genotypicallyand/or phenotypically different from another cell or cell population towhich it is being compared. The “distinct” cells or cell populations mayhowever, be of the same tissue-type; for example, a cancer cell and anon-cancer cell of the same tissue type. In the Example below, lungcancer cell lines (1264, SKLU1, A549 and CALU6) and non-cancer lung celllines (BEAS-2B, CCD19LU and NHBE 4683) were utilized as distinct cellpopulations.

By “selecting” an antibody phage or antibody is meant choosing forfurther analysis, or for employment in further method(s), an antibodyphage or antibody derived therefrom.

An antibody phage or antibody which “binds selectively” to a cell orcell population is one which binds preferentially to that cell or cellpopulation compared to a distinct cell or cell population. The antibodyphage or antibody preferably binds selectively to a cancer cell comparedto a non-cancer cell of the same tissue type. Such selective binding canbe determined by a number of methods known in the art including ELISA(with scFv, Fab or antibody phage); flow cytometry (with scFv, Fab orantibody phage); and immunohistochemistry (with scFv, Fab or antibodyphage).

The act of “counter-selecting” herein refers to binding antibody phagefrom an antibody phage library to a first cell or first cell population(e.g. a non-cancer cell or cell population) which is distinct from asecond cell or second cell population of interest (e.g. a cancer cell orcancer cell population) and subtracting or removing those antibody phagewhich bind to the first cell or cell population (e.g. the antibody phagewhich bind to the first cell or first cell population are not subjectedto subsequent analyses or screening(s)). This may, for example, beachieved by centrifuging antibody phage bound to the first cell(s) andusing the supernatant thereby obtained for further analysis orscreening.

“Expression cloning” refers to the act of characterizing a nucleic acidencoding a protein (e.g. a protein antigen) of interest, wherein themethod involves detecting that protein expressed by the nucleic acid.Detection is possible using an antibody directed against the protein,e.g., an antibody phage or antibody derived from a naïve phage libraryas described herein.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented. The patient to be treated herein may have, or be predisposedto, cancer (e.g. lung cancer). The patient who is “predisposed” tocancer, may display risk factor(s), such as DAF overexpression and/orexpression of a DAF glycoform thought to be associated with cancer.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thedisorder. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXANT™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin (₁ ^(I) and calicheamicin 2^(I)₁, see, e.g., Agnew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin, phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-alpha;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, -beta and -gamma colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; atumor necrosis factor such as TNF-alpha or TNF-beta; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

Drug combinations that are “synergistic” are those in which the combinedaction (e.g. the ability to treat cancer) of the drugs is clinicallysuperior to that of each acting separately.

“Decay Accelerating Factor (DAF)” and “CD55” are used interchangeablyherein and refer to DAF protein as disclosed in U.S. Pat. No. 5,763,224and expressly incorporated herein by reference, including variants andisoforms thereof (see U.S. Pat. No. 5,763,224; Caras et al. Nature 325:545-549 (1987); Lublin et al. J. Immunol. 137:1629-1635 (1986); Hara etal. Immunol. Lett. 37:145-152 (1993); and WO99/43800). Preferred DAF isnative sequence human DAF, including native sequence human secreted DAF(DAF-A) and membrane-bound DAF (DAF-B) (Caras et al. Nature 325: 545-549(1987)). This definition specifically includes glycosylation variants ofDAF, particularly where those variants are preferentially expressed bytumor cells (such as gastric tumor cells, Hensel et al. Cancer Research59:5399-5306 (1999), or lung tumor cells) compared to normal cells ofthe same tissue type. An example of a glycosylation variant is the“791Tgp72 antigen” described in WO99/43800, expressly incorporatedherein by reference.

Examples of antibodies directed against DAF (or antibodies whichspecifically bind to DAF) include the murine monoclonal antibodies IA10,IIH6 and VIIIA7 as described in WO86/07062 published Dec. 4, 1986 andexpressly incorporated herein by reference; the human antibodies hereindesignated LU30, LU13 and LU20; the murine 110 and BRIC 216 monoclonalantibodies directed against DAF as described in WO99/43800; the murine791T36 antibody directed against the 791Tgp72 antigen (ATCC HB9173;WO99/43800); the D17 murine antibody described in Hara et al. Immunol.Lett. 37:145-152 (1993) which binds DAF on blood cells, but not in semenor on testis; the human SC-1 antibody (Vollmers et al. Cancer 76(4):550-558 (1995); Vollmers et al. Cancer Research 49: 2471-2476 (1989);Vollmers et al. Oncology Reports 5:549-552 (1998); and Hensel et al.Cancer Research 59:5299-5306 (1999)), as well as variants of any one ofthe above antibodies. Antibody variants including amino acid sequencevariants (e.g. affinity matured antibodies and humanized variants ofmurine antibodies), glycosylation variants with altered effectorfunction, etc.

A “native sequence” protein comprises the amino acid sequence of aprotein as found in nature, e.g. in a human. The native sequence proteincan be made by recombinant or other synthetic means, or may be isolatedfrom a native source.

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

II. Modes for Carrying Out the Invention

The present application provides a method for making an antibody useful,for example, for cancer diagnosis or therapy, and a method foridentifying an antigen which is differentially expressed on the surfaceof two or more distinct cell populations. These methods employ a naïveantibody phage library that can be prepared according to knowntechniques, including those discussed below.

Antibody Phage Library Preparation

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable regions.Variable domains can be displayed functionally on phage, for example assingle-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).

The naïve repertoire of an animal (the repertoire before antigenchallenge) provides it with antibodies that can bind with moderateaffinity (Kd of about 10⁶ to 10⁷ M⁻¹) to essentially any non-selfmolecule. The sequence diversity of antibody binding sites is notencoded directly in the germline but is assembled in a combinatorialmanner from V gene segments. Each combinatorial rearrangement of V-genesegments in stem cells gives rise to a B cell that expresses a singleVH-VL combination. Immunization triggers any B cells making acombination that binds the immunogen to proliferate (clonal expansion)and to secrete the corresponding antibody. These naïve antibodies arethen matured to high affinity (Kd better than 10⁹ M⁻¹) by a process ofmutagenesis and selection known as affinity maturation. It is after thispoint that cells are normally removed to prepare hybridomas and generatehigh-affinity monoclonal antibodies.

At three stages of this process, repertoires of VH and VL genes can beseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be searched forantigen-binding clones as described in Winter et al., Ann. Rev.Immunol., 12: 433-455 (1994). Unlike libraries from immunized sources, anaïve repertoire can be cloned to provide a single source of humanantibodies to a wide range of non-self and also self antigens withoutany immunization as described by Vaughan et al. Nat. Biotechnol. 14:309-314 (1996); and Sheets et al. PNAS (USA) 95:6157-6162 (1988).Finally, naïve libraries can also be made synthetically by cloning theunrearranged V-gene segments from stem cells, and using PCR primerscontaining random sequence to encode the highly variable CDR3 regionsand to accomplish rearrangement in vitro as described by Hoogenboom andWinter, J. Mol. Biol., 227: 381-388 (1992); and Griffiths et al., EMBOJ., 13:3245-3260 (1994).

Phage display mimics the B cell. Filamentous phage is used to displayantibody fragments by fusion to the minor coat protein pIII. Theantibody fragments can for example be displayed as single chain Fvfragments, in which VH and VL domains are connected on the samepolypeptide chain by a flexible polypeptide spacer, e.g. as described byMarks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments,in which one chain is fused to pIII and the other is secreted into thebacterial host cell periplasm where assembly of a Fab-coat proteinstructure which becomes displayed on the phage surface by displacingsome of the wild type coat proteins, e.g. as described in Hoogenboom etal., Nucl. Acids Res., 19: 4133-4137 (1991). When antibody fragments arefused to the N-terminus of pIII, the phage is infective. However, if theN-terminal domain of pIII is excised and fusions made to the seconddomain, the phage is not infective, and wild type pIII must be providedby helper phage.

The pIII fusion and other proteins of the phage can be encoded entirelywithin the same phage replicon, or on different replicons. When tworeplicons are used, the pIII fusion is encoded on a phagemid, a plasmidcontaining a phage origin of replication. Phagemids can be packaged intophage particles by “rescue” with a helper phage such as M13K07 thatprovides all the phage proteins, including pIII, but due to a defectiveorigin is itself poorly packaged in competitions with the phagemids asdescribed in Vieira and Messing, Meth. Enzymol, 153: 3-11 (1987). In apreferred method, the phage display system is designed such that therecombinant phage can be grown in host cells under conditions permittingno more than a minor amount of phage particles to display more than onecopy of the Fv-coat protein fusion on the surface of the particle asdescribed in Bass et al., Proteins, 8: 309-314 (1990) and in WO92/09690, published Jun. 11, 1992.

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. The use of spleencells and/or B cells or other PBLs from an unimmunized donor provides abetter representation of the possible antibody repertoire, and alsopermits the construction of an antibody library using any animal (humanor non-human) species. For libraries incorporating in vitro antibodygene construction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al., supra and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al., supra or Sastry et al.,supra. Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al., supra, orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorcomformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focussed in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). One canalso make synthetic light chain repertoires (Williams and Winter, Eur.J. Immunol., 23: 1456-1461 (1993)). Synthetic V gene repertoires, basedon a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993); and Griffiths et al.,EMBO J., 13:3245-3260 (1994). The in vivo recombination approachexploits the two-chain nature of Fab fragments to overcome the limit onlibrary size imposed by E. coli transformation efficiency. Naïve VH andVL repertoires are cloned separately, one into a phagemid and the otherinto a phage vector. The two libraries are then combined by phageinfection of phagemid-containing bacteria so that each cell contains adifferent combination and the library size is limited only by the numberof cells present (about 10¹² clones). Both vectors contain in vivorecombination signals so that the VH and VL genes are recombined onto asingle replicon and are co-packaged into phage virions. These hugelibraries provide large numbers of diverse antibodies of good affinity(Kd of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naïve libraries (either natural or synthetic)can be of moderate affinity (Kd of about 10⁶ to 10⁷ M⁻¹), but affinitymaturation can also be mimicked in vitro by constructing and reselectingfrom secondary libraries as described in Winter et al. (1994), supra.For example, mutation can be introduced at random in vitro by usingerror-prone polymerase (reported in Leung et al., Technique, 1: 11-15(1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896(1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). Additionally, affinity maturation can be performed byrandomly mutating one or more CDRs, e.g. using PCR with primers carryingrandom sequence spanning the CDR of interest, in selected individual Fvclones and screening for higher affinity clones. WO 96/07754 (published14 Mar. 1996) describes a method for inducing mutagenesis in acomplementarity determining region of an immunoglobulin light chain tocreate a library of light chain genes. Another effective approach is torecombine the VH or VL domains selected by phage display withrepertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

The antibody phage library of particular interest herein is one whichcomprises from about 10⁹ to about 10¹⁵ antibody phage.

Screening for Useful Antibodies/Antigens

The naïve antibody phage library is panned with or screened against livecancer cells. The cancer cells may, for example, be surgically removedfrom a cancer patient or may be derived from a cancer cell line. Variouscancer cell lines are publicly available, e.g. from the American TypeCulture Collection (ATCC). Exemplary cancer cell lines include breastcancer cell lines such as SK-BR-3, BT-483, MCF-7, BT-20, ZR-751,MDA-MB-231, CAMA1, BT-474; lung adenocarcinoma cell lines such as SKLU1,A549, and 1264; glioma cancer cell lines such as Hs683; ovariancarcinoma lines such as SK-OV-3 and Hey; colorectal carcinoma cell linesincluding HT-29 and Ls180; prostate carcinoma cell lines such as DU145;gastric carcinoma cell lines exemplified by MS; and renal carcinoma celllines such as Caki-1. The cancer from which the cancer cell is derivedmay be a carcinoma, lymphoma, blastoma, sarcoma, or leukemia. Exemplarycancer types from which the cancer cell may be procured include lungcancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, breastcancer, colon cancer, colorectal cancer, salivary gland carcinoma,kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, or hepatic carcinoma. Preferably, the cancer cell is a lungcancer cell.

In a preferred embodiment of the invention, the method includes acounter-selection step using a live non-cancer cell which is preferablyof the same tissue type as the cancer cell. Counter-selection can becarried out at any time including before, during and after (orcombinations thereof) screening the antibody phage library with the livecancer cells. In the preferred embodiment however, counter-selectionprecedes at least one step involving panning against the live cancercells of interest. According to this counter-selection step, the“subtracted” antibody phage (e.g. those present in a supernatant) arethen exposed to the live cancer cells. It was surprisingly discoveredherein that antibodies against an antigen shared by the cancer cell andnon-cancer cell could be identified, in spite of this counter-selectionstep being performed prior to screening the cancer cell of interest. Itwas anticipated that such a counter-selection step may have depleted thephage library of antibodies capable of binding the shared antigen.

The non-cancer cells may, for example, have been surgically removed froma patient or may be obtained from some other in vivo source of thecells, or may be derived from a non-cancer cell line, such cell linesbeing publicly available, e.g., from the ATCC.

The cells to be screened will oftentimes be “adherent” to the extentthey adhere to the surface of a cell culture plate or other solid phasein which they are cultured. The present application provides an improvedmethod for detaching the cells from the surface to which they areadhered comprising the use of a solution which does not include anyprotease and preferably comprises EDTA for detaching the cancer cells.This avoids proteolytic degradation of cell surface antigens resultingfrom the commonly used trypsin release step.

The cancer and non-cancer cells are not fixed prior to exposure toantibody phage in the antibody phage library. Use of such live cellsserves to preserve surface antigens in their native state. Hence, theantibodies prepared according to the present method are more likely tobind the antigen in its endogenous state in a mammal and hence serve assuperior diagnostic (e.g. in vivo diagnostic) and therapeuticantibodies.

Antibody phage from the naïve antibody phage library are contacted with,or bound to, the cancer cells (and optionally the non-cancer cells).Prior to this binding step, an aliquot of antibody phage may be blockedto reduce non-specific binding to cell surfaces. Such blocked antibodyphage may be added to the cells. Alternatively, the cells, which areoptionally blocked, may be added to the antibody phage. The cells andantibody phage are contacted for a sufficient period of time and undersuitable conditions such that binding of the phage to cell surfaceantigen(s) occurs. Such conditions can be determined without undueexperimentation. Moreover, panning steps may be repeated as desired toachieve the desired binding between cell surface antigens and antibodyphage. Cells may be pelleted in-between panning steps via centrifugationor other means as desired.

Binding of antibody phage or antibody derived from the phage to cellsmay, for example, be determined by established methodologies such asELISA, flow cytometry and immunohistochemistry.

Hence, an antibody phage or antibody is selected which binds selectivelyto the cancer cell of interest. Such “cancer-selective” antibodies maybe subjected to one or more further analyses. For example, cloneanalysis (e.g. restriction enzyme cleaving and finger printing and/orDNA sequencing) may be carried out according to known procedures.Alternatively, or additionally, cancer-selectivity of selectedantibodies or antibody phage may be determined by comparing binding ofthe antibodies or antibody phage to cancer cells and non-cancer cells,e.g. of the same tissue type. Such screening may be performed using thecancer and non-cancer cells used to screen the phage library, or othercancer and non-cancer cells.

The selected antibody or antibodies may be altered or modified asdesired to generate an antibody particularly adapted for in vivo therapyor diagnosis. Such alteration may involve one or more amino acidsubstitutions in one or more hypervariable regions of the antibody toincrease its affinity for antigen; i.e. the selected antibody may be“affinity matured”. Moreover, the antibody or affinity matured antibodymay be fused to, or conjugated with, a cytotoxic agent, enzyme (e.g. forADEPT, see below), detectable label, or other antibody (to generate abispecific antibody). Such alterations are discussed in more detailbelow in the Section entitled “Other Methods for Making Antibodies”. Thevariable domain sequences of the antibody or affinity matured antibodymay be fused to human constant region sequences so as to generate alarger antibody molecule, such as a Fab, F(ab′)₂ or intact antibody,depending, for example, on the intended use of the antibody.

Nucleic acid encoding the antibody (which has optionally been altered asexplained in the previous paragraph) may be isolated and inserted into arecombinant expression vector and used to transform a suitable host cellfor expression of the antibody. Exemplary host cells include prokaryotichost cells (e.g. E. coli), yeast cells (such as Saccharomyces cerevisiaeand Pichia pastoris), mammalian cells such as lymphoid cells and ChineseHamster Ovary (CHO) cells, or plant cells. The expressed antibodyrecovered from the host cell, may be used for various diagnostic andtherapeutic applications such as those discussed hereinbelow.

The present method facilitates identification of an antigen expressed athigher levels on a first cell population (generally a cancer cell)compared to a second cell population (e.g. anon-cancer cell of the sametissue type as the first cell). For example, the level of expression ofthe antigen on the first cell population or cancer cell may be about twofold or about five fold to about 100 fold or about 1000 fold greaterthan the level of expression of the antigen on the second cellpopulation or non-cancer cell. Such antigens can be targeted in therapyor diagnosis using antagonists, such as antibodies, or small moleculedrugs directed thereagainst. Antibodies directed against such“over-expressed” antigens can be prepared by screening antibody phagelibraries as discussed above, or according to other methods for makingantibodies available in the art, including those discussed below.

Another advantage of the present invention is the ability to easilyexpression clone nucleic acid encoding the antigen. To expression clonethe antigen, a cDNA library may be prepared, e.g., from the cancer cellused to screen the phage library. The cDNA's thus prepared are expressedin a suitable host cell and expression of the desired protein can bescreened for using one or more antibodies selected from the phagelibrary. This way, cDNA encoding the antigen can be identified andsequenced.

In the present Example, an anti-penta-histidine antibody was used tocross-link, via their penta-histidine epitope tags, scFv fragments usedto screen for expression of desired antigen. This cross-linkingincreased the avidity of the interaction between the scFv and antigen.In addition, an anti-mouse antibody was coated on an assay plate andbound the antibody-linked cells to the assay plate.

Other Methods for Making Antibodies

As disclosed above, the present methods provide means for identifyingantigens expressed at higher levels on one cell compared to another.Such cells may, for example, be cancer cells and the antigen of interestthereon may be one which is useful for targeting with an antibody fortherapy or diagnosis.

Once an antigen is identified as described herein, one can generatefurther antibodies thereagainst by screening antibody phage libraries asdiscussed above, or an antibody can be made by other techniques such asthose disclosed below.

In one embodiment, a polyclonal antibody is raised against the antigenof interest. Polyclonal antibodies are preferably raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 Academic Press (1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63Marcel Dekker, Inc., New York, (1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103Academic Press (1986)). Suitable culture media for this purpose include,for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol,5:256-262 (1993) and Plückthun, Immunol Revs., 130:151-188 (1992).

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Methods for humanizing non-human antibodies are well known in the art.Preferably, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al, J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10: 163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the antigen of interest.Alternatively, an arm which binds antigen of interest may be combinedwith an arm which binds to a triggering molecule on a leukocyte such asa T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG(Fc(R), such as Fc(RI (CD64), Fc(RII (CD32) and Fc(RIII (CD16) so as tofocus cellular defense mechanisms to the cell expressing the antigen ofinterest. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express the antigen. These antibodies possess anantigen-binding arm and an arm which binds the cytotoxic agent (e.g.saporin, anti-interferon-(, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Milstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immuno., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of the antibodyin treating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g. a small molecule toxin or an enzymatically active toxin ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansine (U.S. Pat. No. 5,208,020), a trichothene,and CC1065 are also contemplated herein.

In one preferred embodiment of the invention, the antibody is conjugatedto one or more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per antibody molecule). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) togenerate a maytansinoid-antibody immunoconjugate.

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicinwhich may be used include, but are not limited to, (₁ ^(I), ∀₂ ^(I), ∀₃^(I), N-acetyl-(₁ ^(I), PSAG and 2^(I) ₁ (Hinman et al. Cancer Research53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928(1998)). See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and5,773,001 expressly incorporated herein by reference.

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g. aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

A variety of radioactive isotopes are available for the production ofradioconjugated anti-DAF antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵,Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g. by recombinant techniques or peptide synthesis.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

The antibodies of the present invention may also be used in AntibodyDependent Enzyme Mediated Prodrug Therapy (ADEPT) by conjugating theantibody to a prodrug-activating enzyme which converts a prodrug (e.g. apeptidyl chemotherapeutic agent, see WO81/01145) to an activeanti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as beta-galactosidase andneuraminidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized withbeta-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antibodiesby techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984)).

Diagnostic Methods

The antibody may also be useful in diagnostic assays, e.g., fordetecting expression of an antigen of interest in specific cells,tissues, or serum.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody.

The antibody of the present invention may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibody may also be used for in vivo diagnostic assays. Generally,the antibody is labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C,¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the antigen or cells expressing itcan be localized using immunoscintiography.

Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized antibody formulationsare described in WO 97/04801, expressly incorporated herein byreference. Liquid formulations including antibodies, e.g. as describedin WO98/56418 expressly incorporated herein by reference, are alsocontemplated

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to EGFR, ErbB2, ErbB3, ErbB4, or vascular endothelial factor (VEGF)in the one formulation. Alternatively, or in addition, the compositionmay comprise a cytotoxic agent, cytokine or growth inhibitory agent.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), degradable lactic acid-glycolic acidcopolymers such as the LUPRON DEPOT™ (injectable microspheres composedof lactic acid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid.

Therapeutic Methods

It is contemplated that, according to the present invention, theantibodies may be used to treat various conditions including benign ormalignant tumors (e.g. renal, liver, kidney, bladder, breast, gastric,ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid,hepatic carcinomas; sarcomas; glioblastomas; and various head and necktumors); leukemias and lymphoid malignancies; other disorders such asneuronal, glial, astrocytal, hypothalamic and other glandular,macrophagal, epithelial, stromal and blastocoelic disorders; andinflammatory, angiogenic and immunologic disorders.

The invention provides a method for treating lung cancer (includingsmall-cell lung cancer; and non-small cell lung cancer; e.g. large celllung carcinoma, lung adenocarcinoma and squamous cell lung carcinoma)which comprises administering a therapeutically effective amount of anantibody which is directed against or which specifically binds to DAF,where that antibody is optionally conjugated with, or fused to, acytotoxic agent. The method may also involve co-administering anotheragent useful in treating lung cancer, such as one or morechemotherapeutic agents (e.g. navelbine, gemcitabine, a taxoid,carboplatin, cisplatin, etoposide, cyclosphosphamide, mitomycin orvinblastine) and/or an additional antibody (such as an anti-ErbB2antibody, an anti-angiogenic factor antibody, an anti-mucin antibody, oran antibody directed against a different epitope of DAF, etc) and/orother cytotoxic agent(s) and/or a cytokine. An “angiogenic factor” is agrowth factor which stimulates the development of blood vessels. Thepreferred angiogenic factor herein is vascular endothelial growth factor(VEGF). Such co-administration includes treating with the additionalagent(s) before, simultaneously with (e.g. in one or two separateformulations, or by administering the two or more agents to the patientvia the same IV line, etc), or following, administration of the anti-DAFantibody.

The antibodies of the invention are administered to a human patient, inaccord with known methods, such as intravenous (IV) administration as abolus or by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous administration of the antibody is preferred.

The treatment of the present invention may involve the combinedadministration of an antibody and a chemotherapeutic agent. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers' instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). Thechemotherapeutic agent may precede, or follow administration of theantibody or may be given simultaneously therewith. The antibody may becombined with an anti-estrogen compound such as tamoxifen or ananti-progesterone such as onapristone (see, EP 616 812) in dosages knownfor such molecules.

It may be desirable to also administer antibodies against other tumorassociated antigens, such as antibodies which bind to the EGFR, ErbB2,ErbB3, ErbB4, or vascular endothelial factor (VEGF). Sometimes, it maybe beneficial to also administer one or more cytokines to the patient.

The patient may also be subjected to radiation therapy in conjunctionwith administration of the antibody.

For the prevention or treatment of disease, the appropriate dos age ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of this invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

EXAMPLE 1

In the present example, a large naïve human single chain (sc) Fv phagelibrary was used to search for tumor-associated antigens by panning witha lung adenocarcinoma cell line, 1264, and counter-selecting with anon-tumor bronchial epithelial cell line, BEAS-213. After 3 rounds ofsubtractive panning, 239 out of 673 clones analyzed, bound selectivelyto 1264 tumor cells in a phage ELISA. Diversity analysis of thesetumor-selective clones by BstNI fingerprinting and nucleotide sequencingrevealed 14 distinct scFv fragments. Four clones bound selectively to1264 over BEAS-2B cells when analyzed by a more discriminating flowcytometric assay using scFv. Moreover, these clones showed only limitedcross-reactivity to several primary human cell lines. One clone, LU30,also cross-reacted strongly with the lung adenocarcinoma line, A549. TheLU30 antigen was identified as decay-accelerating factor (DAF, CD55) byexpression cloning from a 1264 cDNA library. The mean number of DAFmolecules on the surface of 1264 and BEAS cells used for panning andcounter-selection were estimated as 75,000±5,000 and 13,000±10,000,respectively. Thus, phage library panning combined with expressioncloning permits identification of antibodies and their cognate antigensfor proteins that are differentially expressed on the surface ofdistinct cell populations.

Materials and Methods

Cell Lines. The lung adenocarcinoma line, 1264, was kindly provided byDr. A. Gazdar, (Simmons Cancer Center, University of Texas-Southwestern,Dallas, Tex.) and grown in RPMI media supplemented with 10% (v/v) FBS.The lung adenocarcinoma cell lines, SKLU1, A549, and CALU6, wereobtained from the ATCC and grown in a 1:1 mixture of RPMI and DMEM mediasupplemented with 10% (v/v) FBS (RPMI/DMEM BS). The BEAS-2B cell line,constructed by SV40 transformation of human bronchial epithelial cells,was obtained from the ATCC, as was CCD19LU, a fibroblast-like cell lineisolated from normal human lung. Both BEAS-2B and CCD19LU cells werecultured in RPMI/DMEM/FBS media. Normal human bronchial epithelial (NHBE4683) and normal human epidermal keratinocytes (NHEK 4021) are primarycell lines (Clonetics, San Diego, Calif.) that were cultured in theserum-free media, BEGM and KGM (Clonetics), respectively. NHBE 4683 andNHEK 4021 lines were used for subtractive panning or analysis betweenthe third and fourth passage. All cell lines were adherent and detachedwith 2.5 mM EDTA in PBS prior to use.

Live Cell Panning with scFv Phage. An aliquot containing 2.5×10¹² cfuphage, from a large human scFv phage library (Vaughan et al. Nat.Biotechnol. 14: 309-314 (1996)) was blocked with 500 :l RPMI containing10% (v/v) FBS, 1 mM PMSF and 2.5 mM EDTA to reduce non-specific bindingto cell surfaces. The blocked phage were added to 1×10⁶ BEAS-2B cells in500 :l RPMI/DMEM/FBS media and mixed gently for 30 min at −20° C. Cellswere then pelleted, at this and subsequent panning steps, bycentrifugation at 500×g for 5 min at 4° C. The phage-containingsupernatant was used to resuspend a fresh pellet of 1×10⁶ BEAS-2B cellsand incubated for 30 min at ˜20° C. followed by pelleting the cells.After repeating this counter-selection step the resultant “subtracted”phage supernatant was incubated with 5×10⁶ 1264 cells for 1 h at −20° C.with gentle mixing. The cells were pelleted and washed 3 times with PBS.The cell-bound phage were eluted with 0.5 ml PBS containing 100 mMcitric acid, pH 2.2 for 10 min and then neutralized with 0.5 ml 1.0 MTris-HCl, pH 7.5.

Escherichia coli strain TCl (New England Biolabs, Beverley, Mass.) inmid-logarithmic growth phase (A₅₅₀=0.4-0.8) was infected with the elutedphage and plated on 2YT agar containing 2% (w/v) glucose and 50 :g/mlcarbenicillin (2YTGC). The resultant colonies were propagated and usedto prepare phage (Marks er al, J. Mol. Biol., 222: 581-597 (1991)). Analiquot containing ˜1×10¹² cfu phage was used for a second round ofpanning consisting of 5 counter-selections using 1×10⁶ BEAS-2B cellsfollowed by selection using 1×10⁷ 1264 cells for ˜15 h at ˜20° C. After10 washes with PBS, the cell-bound phage were eluted and thenneutralized as in the first round of panning. The eluted phage werepropagated and a third round of panning was performed using 1.0×10¹² cfuphage and the second round protocol.

Cell ELISA with Phage. The scFv-phage were compared in their binding tolive tumor and non-tumor cells by ELISA as a primary screen of theirbinding specificity. After the third round of panning a culture of TG1was infected with the eluted phage and plated on 2YTGC. Clones foranalysis were transferred into 96 well plates with 100 :l 2YT mediacontaining 2% (w/v) glucose and carbenicillin (100 :g/ml) and grown for˜18 h with agitation at 30° C. 50 :l 50% (v/v) glycerol was added toeach well of these master plates prior to storage at −70° C.

Replicas of the master plates were prepared and scFv-phage induced bysuperinfection with M13KO7 helper phage and overnight incubation at 30°C. (Marks et al., J. Mot Biol., 222: 581-597 (1991). The plates werecentrifuged (300×g, 5 min, 4° C.) at this and subsequent cell ELISAsteps, to pellet the bacteria and 100 :l scFv-phage containingsupernatants were transferred to 96 well plates containing 100 :l 6%(w/v) bovine serum albumin in PBS per well. 100 :l of the blockedscFv-phage supernatants were added to parallel plates containing either1×10⁵ 1264 or BEAS-2B cells per well (1 h, 4° C., gentle agitation). Theplates were centrifuged and supernatants aspirated without disturbingthe pellets. The cells were washed twice by resuspension in 200 :l 4%(v/v) FBS in PBS (ELISA buffer) at 4° C. followed by centrifugation.Pellets were then resuspended in 100 :l ELISA buffer containing a1:5,000 dilution of horse radish peroxidase conjugated to a sheepanti-M13 polyclonal (Amersham Pharmacia Biotech, Piscataway, N.J.) andincubated for 20 min at 4° C. Cells were centrifuged and washed 3 timesin ELISA buffer. Cell pellets were resuspended in 100 :l TMB reagents(Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.) anddeveloped for ˜10 min prior to quenching with 100 :l 1 M phosphoricacid. The ELISA plates were read (A₄₅₀-A₆₅₀) using a SPECTRAMAX™ 1340microtiter plate reader (Molecular Devices, Sunnyvale, Calif.) and dataanalyzed using a spreadsheet program (Microsoft Excel 5.0a).

Flow Cytometry with Phage and scFv. Culture supernatants containing scFvphage were incubated with cells and washed as described above for thecell ELISA with the following modifications. The anti-M13 polyclonalantibody was used in unconjugated form. After washing, the cells wereincubated for 20 min at 4° C. with an R-phycoerythin-conjugated F(ab′)₂fragment from a donkey anti-sheep IgG (Jackson ImmunoresearchLaboratories, West Grove, Pa.) diluted 1:200 in ELISA buffer, followedby 3 washes and resuspension in 0.5 ml ELISA buffer. Cells were analyzedusing a FAC Scan flow cytometer (Beckton and Dickinson, Mountain View,Calif.).

For cytometric analysis with scFv fragments, 1×10⁵ cells in ELISA bufferwere incubated for 1 h at 4° C. with 3 :g/ml scFv fragment. The cellswere washed twice by centrifugation and resuspension in ELISA buffer.Cell pellets were then resuspended in 100 :l ELISA buffer containing 1:g/ml of the anti-hexahistidine monoclonal antibody, BMG-His1(Boehringer Mannheim, Indianpolis, Ind.). Cells were washed 3 times inELISA buffer before resuspension in 100 :l ELISA buffer containing a1:200 dilution of a F(ab′)₂ fragment of a goat anti-mouse IgG conjugatedwith FITC (Jackson Immunoresearch laboratories). After 3 additionalwashes the cells were analyzed by flow cytometry.

Quantitation of Cell Surface DAF. The mean number of DAF molecules percell was estimated by flow cytometry using a FITC-labeled antibody incomparison with FITC-conjugated beads using the method of Christensenand Leslie J. Immunol. Methods, 132: 211-219 (1990) with the followingmodifications. 250 :g murine anti-DAF monoclonal antibody, 1A10,(Genentech) in 50 mM sodium carbonate, pH 8.5 was incubated with 12 :gN-hydroxysuccinimidyl-fluorescein (Pierce, Rockford, Ill.) for 2 h at20° C., followed by extensive dialysis against PBS. The molar ratio ofFITC to protein was determined from the absorbance at 280 nm and 492 nm(Christensen et al., J. Immunol. Methods, 132: 211-219 (1990)). Cellswere incubated with varying levels of the FITC-labeled anti-DAF antibodyto achieve saturation and then prepared for flow cytometry, as above.

Clone Diversity Analysis. The diversity of antigen-positive clones wasanalyzed by PCR-amplification of the scFv insert using the primers,fdtetseq and PUC19 reverse (Nissim et al., EMBO J., 13:692-698 (1994)),digestion with BstNI (Marks et al., J. Mol. Biol., 222: 581-597 (1991))and analysis by polyacrylamide gel electrophoresis. Comparison of BstNIfingerprints was facilitated by digitization of the gel data using anAlphaImager (Alpha Innotech Corp, San Leandro, Calif.) and analysisusing ProRFLP version 2.34 (DNA ProScan, Nashville, Tenn.). Up to 10clones per BstNI fingerprint were then cycle-sequenced usingrhodamine-labeled dideoxy chain terminators (Applied Biosystems, FosterCity, Calif.), using M13 reverse (New England Biolabs) and mycseq10primers (Nissim et al., EMBO J., 13:692-698 (1994)). Samples wereanalyzed using Applied Biosystems Automated DNA Sequencers (models 373and 377) and sequence data analyzed using the program Sequencher version3.1 (Gene Codes Corp., Ann Arbor, Mich.).

scFv Production. Selected scFv clones were transformed into TG1 andcultured for 18 h at 30° C. in 2YT media containing 0.2 mMisopropyl-∃-D-galactopyranoside to induce scFv expression. Periplasmicextracts were prepared by resuspending a bacterial pellet from a 500 mlculture in 10 ml 50 mM sodium phosphate buffer, pH 8.0 containing 0.5 MNaCl, 25 mM imidazole, 0.2 mg/ml hen egg white lysozyme and 1 mM PMSF.After incubation for 1 h at 4° C. the debris was removed bycentrifugation. Supernatants were filtered (0.2:m) and the His-taggedscFv fragments purified by immobilized metal affinity chromatographyusing Ni²⁺-nitrilotriacetic acid agarose (Qiagen, Valencia, Calif.). ThescFv fragments were eluted with 250 mM imidazole in PBS then dialyzedinto PBS, flash frozen and stored at −70° C. Clones LU1, LU4, LU13,LU20, and LU30 were grown to high cell density in the fermentor aspreviously described (Carter et al., Bio/Technol., 10: 163-167 (1992)).scFv fragments were purified from 2 g fermentation pastes as for cellpellets from shake flasks.

cDNA Library Construction. Total cellular RNA was purified fromguanidine thiocyanate homogenates from 6 g of cultured 1264 cells(Chirgwin et al., Biochemistry, 18: 5294-5299, (1979)). mRNA wasisolated from the total RNA using oligo-d(T) cellulose (CollaborativeResearch, Bedford, Mass.) (Aviv et al., Proc. Natl. Acad. Sci. USA,69:1408-1412 (1972)). Oriented cDNA transcripts were prepared from 5 :gpoly-(A)+ mRNA using the SuperScript Plasmid System (Gibco BRL,Gaithersburg, Md.), fractionated by electrophoresis on a 5%polyacrylamide gel and size selected in the ranges of 0.6-2.0 kb and >2kb. Eluted cDNAs were ligated into the XhoI and NotI sites of themammalian expression vector pRK5 (Suva et al., Science, 237:893-896(1987)), and then electroporated into DH10B (Gibco BRL) cells underconditions recommended by the supplier.

Antigen Expression-cloning from cDNA Library. DNA from 10 pools of50,000 clones each of the 0.6-2 kb and ≧2 kb cDNA libraries was preparedfor expression-cloning the antigens recognized by tumor-selective scFvfragments. 10 :g plasmid DNA from each of the 20 pools waselectroporated into 2×10⁶ COS7 cells in 180 :l PBS using 4 mm gapcuvettes with a Gene Pulser electroporator (BioRad, Hercules, Calif.)with an applied voltage of 300 V and a capacitance of 125 :F. Afterincubation for 72 h at 37° C. the COS7 cells were detached with 2.5 mMEDTA in PBS. The cells were washed and then incubated in 1 ml growthmedia containing one or more purified scFv fragment (10 :g/ml each) for1 h at 4° C. The cells were washed twice to remove unbound scFv,resuspended in 1 ml media containing 5 :g anti-penta-histidine antibody(Qiagen) and incubated for 1 h at 4° C. After 2-3 washes the cells wereresuspended in 5 ml media and transferred to a polystyrene dish coatedwith a polyclonal anti-mouse IgG (ICN/Cappel, Aurora, Ohio) and allowedto bind for 1 h at 4° C. Plates were washed gently 3-4 times with PBS.Remaining attached cells were lysed, plasmid DNA extracted and amplified(Seed et al., Proc. Natl. Acad. Sci. USA, 84: 3365-3369 (1987)). ThisDNA was then electroporated into COS7 cells for additional panning. Inone case, an increasing number of cells were captured during the secondto fourth rounds of panning. Plasmid DNA extracted from the COS7 cellswas transformed into TG1 and single colonies were picked into 96 wellplates. DNA was prepared from pools of 10-20 clones each, electroporatedinto COS7 cells and panned with scFv fragments as described above. Poolsof clones positive for cells binding to the petri dishes were brokendown from the E. coli master plates and individual clones tested bypanning. An individual positive clone was cycle-sequenced usingrhodamine-labeled dideoxy chain terminators.

Affinity Measurements. Kinetic measurements were made by surface plasmonresonance using a BIACORE 1000™ Biosensor (Biacore, AB Uppsala, Sweden).CM-5 chips were functionalized with 350 response units recombinant humanDAF in 10 mM sodium acetate (pH 4.6) or 8,000 response units bovineserum albumin as a negative control. The DAF-derivatized chip wassaturated with LU30 scFv (25-100 nM) by injecting this fragment at 10:l/min in PBS containing 0.5% (w/v) bovine serum albumin and 0.05% (v/v)TWEEN 20™. The resultant sensorgrams were analyzed using BIAEVALUATION™software 3.0.

Results

Subtractive Cellular Panning with scFv Phage. A large human scFv-phagelibrary (Vaughan et al. Nat. Biotechnol. 14: 309-314 (1996)) was used tosearch for novel tumor-associated antigens by panning with the lungadenocarcinoma cell line, 1264, and counter-selecting with the non-tumorbronchial epithelial cell line, BEAS-2B. Precautions were taken tomaintain the integrity of membrane antigens during panning to facilitatesubsequent identification of antigen by expression-cloning usingisolated scFv fragments. Firstly, live rather than fixed cells were usedfor panning in an attempt to preserve surface antigens in their nativestate. Secondly, cells grown adherently were detached with EDTA alone,thereby avoiding proteolytic degradation of cell surface antigensresulting from the commonly used trypsin release step.

The number of phage recovered after 1, 2 and 3 rounds of panning was1.5×10⁷, 7.0×10⁵ and 4.0×10⁶ cfu, respectively. The phage populationsafter each round of panning were analyzed by flow cytometry. The phagefrom the third round showed a large increase in binding to 1264 cellsand a slightly smaller increase with BEAS-2B cells when compared tophage from prior rounds and unselected phage (FIG. 1). This apparentdifferential increase in binding to 1264 over BEAS-2B cells encouragedus to screen individual phage from the third round population forselective binding to the 1264 tumor cells.

Analysis of Clone Specificity and Diversity. The binding specificity ofindividual clones from the third round of panning was analyzed by ELISAusing scFv-phage and live cells. The primary criteria used to assesstumor-selectivity were robust binding to 1264 cells (ELISA signal:A450-A₆₅₀≧0.3) and much weaker if detectable binding to BEAS-2B cells(≧20-fold lower ELISA signal). The diversity of clones satisfying theseprimary criteria was assessed by BstNI fingerprinting of thePCR-amplified scFv fragments, and nucleotide sequencing of up to 10clones per fingerprint pattern. A small number of clones that did notsatisfy the primary criteria were also fingerprinted (n=29) andsequenced (n=11). Secondary criteria were then used to chose unique andapparently tumor-selective clones for further analysis: 1) unambiguousfingerprint pattern, 2) open reading frame for scFv, 3) majority ofclones which share the same nucleotide sequence also satisfy the primaryselection criteria.

Out of 673 clones analyzed, 239 satisfied the primary criteria forselective binding and 197 clones could be assigned to 15 different BstNIfingerprint patterns (Table 1). In the majority of cases (13/15) onefingerprint pattern gave rise to a single nucleotide sequence, whereasin 2/15 cases 2 different sequences were found with indistinguishableBstNI fingerprint patterns. Thus a total of 17 scFv clones wereidentified that satisfy the secondary selection criteria. The 2 mostabundant clones, fingerprints types 1 and 2, represented ˜80% of theclones satisfying the secondary criteria. In contrast, the other 15clones each represent ≧5% of the clones identified. Four of the 17clones (LU1, LU3, LU22 and LU36) are very closely related (≧97% aminoacid identity for scFv) (FIG. 2A). Thus from the 673 clones initiallyscreened, 14 distinct scFv clones were identified that show selectivebinding to BEAS-2B cells as judged by phage ELISA. These 14 distinctscFv fragments have divergent V_(H) sequences (FIG. 2B) whereas theircorresponding V_(L) domains are more limited in diversity (FIG. 2C).Indeed, many of the scFv clones isolated utilize identical or veryclosely related V_(L) sequences as previously noted (Vaughan et al. Nat.Biotechnol. 14: 309-314 (1996); Merchant et al. Nat. Biotechnol. 16:677-681 (1998)). This reflects the very limited size of the light chainrepertoire in the phage library.

Stringent Analysis of Clone Specificity. The 14 distinct clones thatsatisfied the secondary selection criteria (Table 1) were furtheranalyzed by a more discriminating but low throughput flow cytometricscreen using scFv fragments (representative data in FIG. 3). Fourclones, namely LU1, LU13, LU20 and LU30, demonstrated significantbinding to 1264 cells but minimal cross-reactivity to BEAS-2B cells. Incontrast, the remaining clones, (represented by LU4 in FIG. 3) showedsignificant cross-reactivity to BEAS-2B. Clone LU30, which gave the mostpronounced binding to 1264, also gave strong staining of 1/3 additionallung adenocarcinoma lines tested (A549). Clones LU1, LU13, LU20 and LU30showed minimal cross-reactivity to BEAS-2B and the primary human line,NHEK 4021 (FIG. 3). Flow cytometric analysis with the primary humanlines, CCD-19LU and NHBE 4683, gave very similar binding between LU1,LU13, LU20 and LU30 phage as control phage, albeit with substantiallyhigher background binding than for the other lines.

Expression Cloning of LU30 Antigen. ScFv fragments corresponding toclones LU13, LU20 and LU30 were prepared by secretion from E. coli andimmobilized metal affinity chromatography and used for expressioncloning. Panning was performed using a mixture of these 3 scFv fragmentsand a cDNA expression library constructed from 1264 cells that wastransiently expressed in COS7 cells. After 3 rounds of panning using amixture of these 3 scFv fragments, efficient cell capture wasdemonstrated with some plasmid pools using LU30 but not LU13 and LU20scFv fragments. Repeated panning using clones LU13 and LU20 in theabsence of LU30 was also unsuccessful. Positive pools for clone LU30were broken down first into smaller pools and then into individualclones. This led to the identification of a single clone expressing aprotein that bound specifically to the LU30 scFv fragment. Nucleotidesequence analysis of this clone identified it as decay acceleratingfactor (DAF, CD55). Binding of LU30 scFv (GenBank accession numberAF117206) to 1264 cells could be competed with recombinant human DAF(FIG. 4) but not with the anti-DAF monoclonal antibody, 1A10 (data notshown). Further confirmation of LU30 binding to DAF was provided byaffinity measurements obtained using a BIACORE™ instrument: K_(d)=(13±5)nM, k_(on)=(3.4±1.0)×10⁵ M⁻¹s⁻¹, k_(off)=(4.5±1.3)×10⁻³ s⁻¹.

Cellular DAF Levels. The mean number of DAF molecules on 1264 andBEAS-2B cell lines was estimated by quantitative flow cytometry using ananti-DAF IgG labeled with a mean number of 5.3 FITC molecules incomparison with standards. The number of DAF molecules on the 1264 tumorcells used for panning and BEAS non-tumor cells used forcounter-selection were estimated as 75,000±5,000 and 13,000±10,000,respectively. Attempts to estimate the number of DAF sites on BEAS-213and 1264 using this methodology with the LU30 scFv fragment wereunreliable since FITC labeling of LU30 scFv impaired its binding to DAF.

Discussion

Four scFv fragments were identified that bound more extensively to oneor more tumor cell lines than to related non-tumor cell line(s) bysubtractive panning of live cells with a large naïve antibody phagelibrary. The cognate antigen corresponding to one scFv clone, LU30, wasidentified as DAF by expression cloning. DAF is expressed atapproximately 6-fold greater levels on 1264 cells than BEAS cells usedfor counter-selection. Thus the counter-selection process is not 100%efficient, permitting identification of a scFv fragment that binds toantigen that is present at much higher levels on target than controlcells. This bodes well for the utility of this method since cell surfaceantigens that are overexpressed in tumors compared to normal tissuesoccur frequently, e.g. HER2/neu (Tzahar et al., Biochim. Biophys. Acta,1377:M25-M37 (1998)) and EGFR (Voldborg et al., Annals Oncol.,8:1197-1206 (1997)).

Antibody phage panning method offers a potential direct and broadlyapplicable route to the identification of human antibodies suitable foranti-tumor therapy. This strategy likely favors the identification ofantibodies to highly expressed antigens, such as DAF shown here, sincehigh antigen levels are anticipated to facilitate enrichment ofcognate-scFv phage during panning. This seems desirable since high levelantigen expression may also facilitate tumor localization of anti-tumorantibodies in vivo.

Antibody phage panning could potentially identify tumor-associatedantigens resulting from post-translational modifications that differbetween tumor and non-tumor cells, e.g., the mucin product of the MUC1gene is underglycosylated in many human tumors (Barratt-Boyes et al.,Cancer Immunol. Immunother., 43:142-151 (1996)) exposing new epitopesfor antibody targeting. This has prompted the development of humanizedanti-MUC1 antigen (Couto et al., Adv. Exp. Med. Biol., 353:55-59 (1994);Couto et al., Hybridoma, 13:215-219 (1994); Baker et al, Adv. Exp. Med.Biol., 353: 61-82 (1994)). Furthermore human antibodies recognizing MUC1on tumor cells have been identified by panning with a MUC1 peptide(Henderikx et al. Cancer Res. 58: 432-44332 (1998)). In contrast, suchpost-translational differences between tumor and non-tumor cells willnot be detected by powerful high throughput transcriptome and genomicmethods, such as differential display (Liang et al., Curr. Opin.Immunol. 7: 274-280 (1995)) cDNA (Schena, M. et al., Science,270:467-470 (1995); DeRisi et al., Nat. Genet., 14:457-460 (1996)) oroligonucleotide (Chee et al. Science, 274:610-414 (1996)) microarray andSAGE (Velculescu et al., Science, 276:1268-1272 (1997); Zhang et al.Science, 276:1268-1272 (1997);

Hibi et al., Cancer Res., 58: 5690-5694 (1998)). Transcriptome andgenomic methods will also fail to detect proteins which areoverexpressed in tumors despite unchanged RNA transcript levels and genecopy number, respectively.

SAGE has identified significant differences in RNA transcript levelsbetween primary human tumors and tumor cells lines (Zhang et al.Science, 276:1268-1272 (1997)). This raises the possibility thatantibody phage panning may detect tumor-associated antigens found onprimary human tumors but not cell lines. Conversely antibodies may beidentified that are cell line specific as judged by failure to bindprimary human tumor cells. Direct panning on primary human tumor cellsis anticipated to avoid these problems.

As judged by immunoaffinity purification followed by western blottingwith the anti-DAF monoclonal antibody IA10 (WO86/07062), LU20 and LU13were also found to bind to DAF. The VL and VH sequences of the LU30,LU20 and LU13 antibodies are shown in FIGS. 5A and 5B.

EXAMPLE 2

This Example describes how one may treat a human patient with lungcancer with a human antibody as described herein.

The VH and VL domains of the human antibody LU30 identified as describedin the previous Example are joined to human IgG1 constant domains togenerate an intact antibody with effector functions in vivo. Theantibody may be expressed in a Chinese Hamster Ovary (CHO) cell (U.S.Pat. No. 4,816,567, expressly incorporated herein by reference). Therecombinant antibody is recovered from the CHO cells and formulated as alyophilized preparation which can be reconstituted with bacteriostaticwater for injection (BWFI) to generate a reconstituted formulation forintravenous or subcutaneous administration to a human patient (see WO97/04801). The reconstituted formulation is administered to a humanpatient diagnosed as having lung cancer, e.g. in an initial loading doseof about 4 mg/kg IV followed by weekly doses of about 2 mg/kg IV.Candidate patients for therapy may optionally be screened to determinewhether they express variant OAF (e.g. a glycosylation variant of DAF)which is preferentially expressed on cancerous lung tissue as opposed tonormal (i.e. noncancerous) lung tissue and/or to establish whether theirtumor overexpresses DAF. Immunohistochemistry and DNA-based assays (e.g.fluorescent in situ hybridization, FISH) that can be used to determinegene amplification and/or protein overexpression are readily availablein the art. The human antibody, LU30, may for instance be used to assessDAF overexpression via IHC. The anti-DAF antibody is optionally combinedwith other cytotoxic agents used to treat lung cancer, such asnavelbine, gemcitabine, a taxoid, carboplatin, cisplatin, etoposide,cyclosphosphamide, mitomycin, vinblastine and/or an additional antibody(such as an anti-ErbB2 antibody, anti-angiogenic factor antibody, ananti-mucin antibody, or an antibody directed against a different epitopeof DAF) in amounts conventially used for such agents. Administration ofthe anti-DAF antibody to the patient is anticipated to increase the timeto disease progression, result in higher overall response rates (ORRs),increase the median duration of response and/or increase 1-year survivalrate compared to placebo-treated patients.

1. A method for making an antibody against an antigen which is shared bya cancer cell and a non-cancer cell comprising the following steps: (a)counter-selecting antibody phage from a naïve antibody phage libraryusing a live non-cancer cell; (b) subsequently binding the antibodyphage to a live cancer cell; (c) selecting an antibody phage or antibodywhich binds selectively to the live cancer cell; and (d) identifying anantigen to which the antibody phage or antibody binds.
 2. The method ofclaim 1 wherein the mean number of antigen molecules per cancer cell isgreater than the mean number of antigen molecules per non-cancer cell.3. The method of claim 2 wherein the mean number of antigen moleculesper cancer cell is about two fold to about 1000 fold greater than themean number of antigen molecules per non-cancer cell.
 4. The method ofclaim 2 wherein the mean number of antigen molecules per cancer cell isabout five fold to about 100 fold greater than the mean number ofantigen molecules per non-cancer cell.
 5. The method of claim 1 whereinthe non-cancer cell is of the same tissue-type as the cancer cell. 6.The method of claim 1 wherein the cancer cell is a lung cancer cell. 7.The method of claim 1 wherein the antibody phage library comprises fromabout 10⁹ to about 10¹⁵ antibody phage.
 8. The method of claim 1 furthercomprising expression cloning the antigen.
 9. The method of claim 1wherein the cancer cell is from a cancer cell line.
 10. The method ofclaim 1 further comprising comparing binding of the antibody phage orantibody to a cancer cell and a non-cancer cell.
 11. The method of claim1 further comprising detaching the cancer cell from a surface to whichthe cancer cell is adhered using a solution which does not include anyprotease.
 12. The method of claim 1 wherein the solution comprises EDTAfor detaching the cancer cell.